Many viruses of the serologically related insect-borne flavivirus genus are important human pathogens, including yellow fever virus, Japanese encephalitis virus, tick-borne encephalitis virus, West Nile virus and dengue virus. Among them, the dengue viruses (serotypes 1-4) are most important in terms of human morbidity, which is estimated to involve millions of individuals each year mostly in the tropical and subtropical areas. A safe and effective vaccine against dengue is currently not available. A DEN4 deletion mutant that had been constructed and characterized in this laboratory was evaluated for safety and immunogenicity in human volunteers. Results indicated that this live dengue type 4 virus vaccine was well tolerated and did not cause serious illness in 20 healthy adults. After a single dose all volunteers sero-converted with a 7-fold or greater increase in serum neutralizing antibody titer. However, a mild asymptomatic macular rash developed in 10 volunteers and a transient elevation in serum liver enzyme (ALT) level was found in 5 volunteers. This indicates that this live vaccine candidate is not sufficiently attenuated. Passive immunization using dengue virus neutralizing antibodies provides an attractive alternative to vaccine for prevention of dengue virus infection. Toward this goal, we initiated a new project to identify and characterize antibodies of chimpanzees or humans that effectively neutralize each of the four dengue virus serotypes. We employed the technique of phage display of combinatorial antibody, which has provided a powerful tool for isolation of human or chimpanzee antibodies to important viral pathogens. Two chimpanzees that had been intrahepatically inoculated with DEN4 RNA were sero-converted and each developed a high titer of dengue virus neutralizing antibodies. A combinatorial antibody library was first constructed from the bone marrow lymphocytes of the chimpanzees. Panning of the phage library and subsequent screening have allowed identification of four distinct groups of Fab antibodies reactive to DEN4 on the basis of their sequences in the heavy chain variable region. Three of these Fabs co-precipitated DEN4 PrM and E, whereas the fourth Fab precipitated PrM alone. Competition ELISA showed that two of these Fabs reacted with overlapping sites on E, whereas the other two Fabs reacted with overlapping sites on PrM. Each of these Fab clones weakly neutralized DEN4 in vitro only in the presence of an anti-Fab antibody, suggesting that a multivalent form of these antibodies was required for the neutralizing activity. High level expression of the full-length IgG molecules in mammalian cells should allow determination if any of the antibodies from the Fab clones is capable of neutralizing DEN4. As predicted, a computer search revealed that these chimpanzee Fabs shared a high sequence homology with the human IgG homologs. These are the first monoclonal antibodies specific to DEN4 from higher primates (chimpanzees). To expand the antibody repertoire, another combinatorial antibody library was constructed from chimpanzees hyper-immunized with each of the four dengue viruses. This new antibody library should prove valuable for identification of neutralizing antibodies against each of the four dengue viruses for use in immunoprophylaxis.